Objective:To investigate the distribution of integrons and plasmid-mediated quinolone resistance (PMQR) genes in clinical isolates of Klebsiella aerogenes and to analyze the relationship between integrons and bacterial resistance to antimicrobial agents. Methods:Ninety-one Klebsiella aerogenes strains isolated from clinical samples in the Fengxian District Central Hospital from November 2015 to March 2021 were used in this study. Class 1 and class 2 integron-integrase genes ( intI1 and intI2) and PMQR genes were screened by PCR. The types of promoters and gene cassette arrays of variable regions were determined by sequencing. Besides, the relationship between integrons and antimicrobial resistance was analyzed. Results:The resistance rate of the 91 Klebsiella aerogenes isolates to aztreonam was more than 40.00% and the resistance rates to other commonly used antimicrobial agents were less than 35.00%. Among the 91 isolates, 30 carried the intI1 gene, while none of them carried the intI2 gene. Seven class 1 integron gene cassette arrays of variable regions were detected and the gene cassette array of aac(6′)-11 C- ΔereA2- IS1247- aac3- arr- ΔereA2 was detected in Klebsiella aerogenes. PcH1 with weak activity was the predominant variable region promoter of class 1 integrons. The detection rates of intI1-positive and intI1-negative isolates in ICU, neurosurgery and other clinical departments were statistically different ( P<0.05). The resistance rate of intI1-positive isolates to some commonly used antibiotics was significantly higher than that of intI1-negative isolates ( P<0.05). qnrS gene was the prevalent PMQR gene. The detection rates of integrons and PMQR genes in Klebsiella aerogenes isolates was low except for the strains isolated in 2016. Conclusions:Antimicrobial resistance in Klebsiella aerogenes was closely related to integrons. The distribution of integrons in Klebsiella aerogenes strains isolated from different clinical departments was different, and the monitoring of drug-resistant strains should be strengthened in ICU and neurosurgery. The resistance to quinolones in Klebsiella aerogenes strains in this region was mainly related to qnrS gene.

Objective:To explore the distribution of integrons in Escherichia coli isolated from community patients with urinary tract infections and their relationship with the phylogenetic groups and antimicrobial resistance. Methods:From November 2015 to December 2018, 152 isolates of E. coli that collected without repetition from the urine samples of outpatients in nephrology of Fengxian District Central Hospital in Shanghai, were studied retrospectively. Bacterial identification and antimicrobial susceptibility analysis was carried out by Phoenix 100 automatic microbiological analyzer. Class 1, 2 integron integrase genes, variable regions of integrons and the phylogenetic groups of isolated E.coli were screened by PCR. The type of promoters and gene cassette arrays of variable regions were determined by sequencing. The relationship of intergon with the phylogenetic groups and antimicrobial resistance was also analyzed. Results:The resistance rate of 152 E. coli to ampicillin was 70.39% (107/152), and the resistance rates to other antibacterial drugs were all less than 40.00%. Among the 152 E. coli isolates, class 1 integron integrase gene intI1 was detected in 65 isolates (42.76%), 8 gene cassette arrays and 14 antimicrobial resistance gene cassettes were detected in 68 class 1 integrons. The most popular gene cassette array was dfrA17-aadA5 (51.47%, 35/68), while the variable regions of class 1 integrons were failed to detected in 12 intI1-positive isolates. Five variable region promoters were detected in 68 class 1 integrons, with the relative weak promoter PcH1 to be the most popular type (77.94%, 53/68). The gene cassette array arr- 2-cmlA5-bla OXA-10-aadA1 was also detected in this study. 65 intI1-positive isolates were mainly belonged to group B2 and D. The class 2 integron integrase gene intI2 was detected in 4 isolates (2.63%,4/152), and their variable region gene cassette arrays were all dfrA1-sat2-aadA1. Conclusions:Class 1 integrons were closely related to antimicrobial resistance in E. coli isolated from community patients with urinary tract infection. Most of the variable region promoters of class 1 integrons were relatively weak promoters. The distribution of each phylogenetic group in the intI1-positive isolates was consistent with the distribution of the overall isolates. The gene cassette array arr-2-cmlA5-bla OXA-10-aadA1 was detected in E. coli.

Objective To investigate the value of real-time fluorescent quantitative polymerase chain reaction (RT-qPCR) in the detection of Streptococcus agalactiae in early screening of Streptococcus agalactiae in pregnant women in the third trimester, and to study the effect of Streptococcus agalactiae infection on them.Methods A retrospective analysis of 5 855pregnant women with vaginal discharge and rectal secretions from January 2017to January 2018in our hospital were performed to detect Streptococcus agalactiae by RT-qPCR.Meanwhile, 100vaginal non-vaginosis women in the third trimester were selected as the negative group and100healthy women as the control group.The vaginal micro-environment-related indexes (Lactobacilli content, flora diversity and cleanliness) of three groups were analyzed.Results The total carrier rate of Streptococcus agalactiae of the third trimester from January 2017to January 2018in our hospital was 9.6％, of which vaginal carriage rate was 2.4％, rectum carriage rate was 7.2％, Streptococcus agalactiae positive group (GradeⅡ-Ⅲ), rates of population diversity (GradeⅡ-Ⅲ) cleanliness (Ⅰdegree) and microenvironment imbalance were 30.1％, 36.4％, 25.9％and 76.2％respectively, while those in the negative group were 58.0％, 55.0％, 61.0％and 63.0％and those in the control group were 87.0％, 91.0％, 83.0％, 24.0％.The positive rate of Streptococcus agalactiae in each group was significantly higher than that in the control group (P＜0.05).The differences of all the indexes between the negative group and the control group were also statistically significant (P＜0.05).Conclusion The carrying rate of Streptococcus agalactiae in third trimester pregnancy in our hospital was within the normal range reported in the literature.The detection of Streptococcus agalactiae by RT-qPCR could be effectively screened and monitored.Carrying Streptococcus agalactiae might increase the risk of vaginal infection in third trimester women.

Objective To investigate the diagnostic value of hypersensitive troponin I (hs-cTnI) , homocysteine (Hcy) , hypersensitive C-reactive protein (hs-CRP) and calcitonin (PCT) detection in acute exacerbation of chronic obstructive pulmonary disease (AECOPD) .Methods Ninety-six patients with AECOPD were enrolled in this study.50healthy subjects were included in the healthy control group.Hs-cTnI was measured by electrochemiluminescence, Hcy level was measured by circulating enzyme method, hs-CRP level was determined by immunoturbidimetry, and colloidal gold blotting was performed.PCT levels, correlation analysis using Pearson correlation analysis, analysis of the correlation between the indicators and AECOPD, using ROC curve analysis of hs-cTnI, Hcy, hs-CRP, PCT diagnosis COPE acute exacerbation curve area (AUC) .Results The levels of hs-cTnI, hs-CRP and PCT in the AECOPD group were higher than those in the healthy control group (P<0.05) .The Hcy in the AECOPD group was lower than that in the healthy control group.The difference was not statistically significant (P>0.05) .Pearson correlation analysis shows that hs-cTnI, hsCRP and PCT were positively correlated with AECOPD (r=0.346, 0.401, 0.509) , and Hcy had a poor correlation with AECOPD (r=0.078) .In the ROC curve analysis, the area under the curve (AUC) for hs-cTnI, hsCRP, and PCT were 0.825, 0.834, and 0.922, respectively, The best diagnostic cut-off values were:0.082, 18.25, 3.075, and the sensitivities were 0.823, 0.802, and 0.781, respectively.The specificities were:0.92, 0.62, and 0.94.Youden′s index was 0.743, 0.422, and 0.721, respectively.The kappa values are:0.699, 0.423, 0.664.Conclusion The detection value of Hcy in the diagnosis of AECOPD is low.The detection of hs-cTnI, hsCRP and PCT has a certain diagnostic value in AECOPD.It can be used as an auxiliary diagnosis index of AECOPD.

Molecular testing are more and more widely used in clinical practice. The premise of clinical application is the verification or validation of the molecular testing procedure. Establishing a verification and validation model suitable for the testing procedures of clinical molecular testing is helpful for the practitioners of clinical molecular testing to carry out the verification and validation work more conveniently and efficiently. It is also helpful for the standardization of testing and quality control significantly. This article will elaborate on the status of verification and validation of molecular testing projects, the requirements of the ISO15189 accreditation system, the establishment of verification validation models and the prospects in the field.

Objective To investigate the distribution of integrons in clinical isolates of carbapen-em-resistant Acinetobacter baumannii and their relationships to bacterial resistance to antimicrobial agents.Methods A total of 115 carbapenem-resistant Acinetobacter baumannii strains were isolated from clinical samples of patients from January to October, 2017. Phoenix 100 automatic microbiological analyzer was used for antimicrobial sensitivity analysis. Classes 1 and 2 integrase genes and carbapenemase-encoding genes, bla IMP , blaVIM , blaKPC , blaNDM and blaOXA-23 , were screened by PCR. The variable regions of integrons were amplified by long fragment PCR. The types of promoters and gene cassette arrays of variable regions were de-termined by sequencing and overlap PCR. Relationships between integrons and antimicrobial resistance were analyzed. Results The 115 isolates of carbapenem-resistant Acinetobacter baumannii were resistant to most commonly used antimicrobial agents, but sensitive to polymyxin E. All of the isolates carried blaOXA-23 gene and none of them were positive for blaIMP , blaVIM , blaKPC or blaNDM gene. Class 1 integrase gene intI1 was de-tected in 40 isolates (34. 8％ ), while class 2 integrase gene intI2 was not detected. Two gene cassette ar-rays of variable regions, aacA4-catB8-aadA1 (39 isolates) and aacC1-gacP-gacQ-aadA1a (23 isolates), were detected in intI1-positive isolates. Twenty-two isolates carried both aacA4-catB8-aadA1 and aacC1-gacP-gacQ-aadA1a. The upstream promoters of the variable regions were relatively strong promoters, PcH2 and PcS. The gene cassettes of the variable regions endowed bacteria with resistance to chloramphenicol and aminoglycoside antibiotics. The resistance rate of class 1 integron-positive isolates to compound sulfamethox-azole was higher than that of negative strains. However, their resistance rate to ampicillin/sulbactam was lower than that of negative strains. Conclusions Antimicrobial resistance in carbapenem-resistant Acineto-bacter baumannii was serious. Carbapenem resistance was associated with blaOXA-23 gene. The types of pro-moters of variable regions in class 1 integrons were all relatively strong promoters. Class 1 integrons were closely related to sulfonamides resistance.

Molecular diagnosis plays more and more important role in disease diagnosis, prognosis evaluation and curative effect monitoring.With the support of relevant national policies and the gradual release of accurate medical needs from society, molecular diagnosis relies on its advantages of accuracy, convenience,sensitivity, non-invasiveness and automation to develop rapidly in the direction of precision inspection.The application of molecular diagnostic automation is undoubtedly the catalyst for the development of molecular diagnostics, but its application status is far from satisfactory.It is necessary to analyze the causes,study countermeasures and look forward to the future.

Objective To analyze the structures of atypical class 1 integrons in clinical Proteus isolates. Methods This study included 26 class 1 integron integrase gene-positive clinical Proteus isolates, from which the variable regions of class 1 integrons could not be amplified. Six isolates were chosen from them to amplify the flanking DNA segments of class 1 integron integrase gene using inverse PCR. The se-quences of PCR products were analyzed with BLAST to identify the target homologous sequences as well as their accession numbers in GenBank. Primers for overlap PCR were designed according to the flanking se-quences. Then the 26 clinical Proteus isolates were analyzed with overlap PCR and sequencing analysis. Re-sults The variable regions of class 1 integrons in 25 out of the 26 clinical Proteus isolates were completely or partly identified by using inverse PCR,overlap PCR and sequencing analysis. A gene cassette array of estX-psp-aadA2-cmlA1-aadA1a-qacI-tnpA-sul3 was detected in 22 isolates. The variable regions of class 1 integrons in the other three isolates were identified to be estX-psp-aadA2-cmlA1,dfrA14 and IS26,respectively. All of the 25 isolates lacked the 3'conserved segements in class 1 integron. Conclusion Inverse PCR can be used to analyze the structures of atypical class 1 integrons. Gene cassette psp and gene cassette arrays of estX-psp-aadA2-cmlA1-aadA1a-qacI-tnpA-sul3 and estX-psp-aadA2-cmlA1 in clinical Proteus isolates are reported for the first time.

Objective To understand the infection status and distribution characteristics of human papillomavirus (HPV)in fe-male genital tract in Fengxian district of Shanghai City in order to provide preliminary recommendations for prevention and treat-ment of HPV in this area.Methods The women aged over 1 6 years old receiving HPV testing in the Southern branch Hospital of Shanghai Sixth People′s Hospital From January 2011 to December 2015 were taken as the research subjects and their cervical secre-tions were performed the HPV 21 subtypes detection,among them,1 374 cases underwent colposcopic fixed point biopsy of cervix for determining the pathological grade.Then the data were recorded and analyzed by the SPSS20.0 software.Results Twenty-one HPV subtypes were detected,in which the HPV positive rate was 17.54% (6 313/35 977),and types with high HPV infection rate in these five years were in turn HPV18,HPV52,HPV16,HPVCP8304,HPV58,HPV53,HPV53,HPV53 and HPV58;the HPV positive rate in 1374 cases of cervical lesions was 82.75% (237/1 374),the common types distributions in cervical lesions were HPV16,HPV58,HPV52,HPV18 and HPV31 respectively.Conclusion The HPV infection situation in Fengxian district of Shang-hai City has certain regional characteristics.Then the HPV prevention and treatment strategy suitable for this region may be formu-lated according to the research results.

Objective To develop a simple high-resolution melting ( HRM) analysis method for differentiation of Pc and P2 variants in class 1 integron.Methods DNA fragments containing Pc and P2 variants were amplified from plasmids pACW ( PcW ) and pACWP2 ( PcW-P2 ) respectively , then these purified PCR products and P 2 promoters were analyed full-length amplicon by HRM .Eight DNA fragments containing different Pc promoters were amplified and site-specific mutated from plasmids pACS ( PcS ) , pACH2 ( PcH2 ) , pACH1 ( PcH1 ) , pACW ( PcW ) , genomic DNA of Klebsiellar pneumonia HS07-68 (PcWTGN-10)and HS05-1792(PcH2TGN-10)respectively.The purified PCR products and eight Pc variants were characterized by HRM analyses of an unlabeled probe and full-length amplicon.This assay was applied to the differentiate Pc and P2 variants in 109 class 1 integrons from 95 urine clinical Escherichia coli isolates in Huashan Hospital during 2004 -2007.The differentiation results were compared with that determined by direct sequencing .Results P2 promoter with a significant higher melting temperature ( Tm ) can be identified by HRM analysis clearly .P2 promoters were identified in 2 class 1 integrons and consistent with direct sequencing results .Eight Pc variants were classified into three groups: PcS, PcSTGN-10 , PcW, PcWTGN-10, PcH1, PcH1TGN-10.Using direct HRM analysis.PcH2, PcH2TGN-10 were classified into four groups:PcS, PcH1, PcH2, PcW, PcSTGN-10 , PcH1TGN-10 , PcH2TGN-10 , PcWTGN-10 according to the melting curves of the unlabeled probe .Combined the HRM analyses of the whole amplicon and unlabeled probe , the eight Pc variants can be differentiated from each other .Five different Pc variants, PcS, PcW, PcH1, PcH2TGN-10 and PcWTGN-10 , were identified and consistent with direct sequencing results .Conclusions This developed a simple Pc and P 2 variants differentiation method via simultaneous HRM analyses of an unlabeled probe and full-length amplicon .This method is cost-effective and accurate , could be used in differentiation of Pc and P2 variants of class 1 integrons in clinical isolates .